Biochemistry and genetics of the ORMDL regulators of sphingolipid biosynthesis

鞘脂是一种多样但代谢相关的脂质组，具有独特的功能和物理性质， 对细胞信号传导和膜结构至关重要的性质。特别是监管不当 鞘脂与多种疾病如癌症、炎性疾病和 心血管疾病特别是，如下所述，全基因组关联研究强烈地 暗示这个提议的主题，鞘脂代谢调节剂ORMDL，在儿童的风险 哮喘细胞鞘脂的总体水平由细胞内的起始酶和限速酶决定。 生物合成途径，丝氨酸棕榈酰转移酶（SPT）。毫不奇怪，SPT活动是稳态的， 受控-即SPT活性被升高水平的细胞鞘脂强烈抑制。我们有 证明了这种稳态控制是由一组小的膜蛋白介导的，这些蛋白存在于细胞膜中。 内质网，ORMDLs。本文提出的研究目标是通过以下方式建立该机制： ORMDLs介导的SPT抑制对鞘脂水平升高的反应，并开始揭示 这些机制如何影响哮喘的风险。这些研究将回答基本问题， ORMDLs感测细胞鞘脂水平以及鞘脂水平的变化如何通过 ORMDL影响SPT活性。我们将检验神经酰胺直接结合ORMDL触发SPT的假设 调控我们将确定ORMDL中的结构元件，这些结构元件对于介导鞘脂 调节SPT，将使用最近构建的无细胞系统来定量鞘脂水平 通过ORMDL系统感知，并将测试鞘脂升高诱导神经元内分泌变化的假设。 ORMDL拓扑。最后，我们将用纯化的ORMDL和SPT构建一个重组系统， 假设ORMDLs和SPT是该调节系统的唯一必需组分。此外 我们建议解决一个关于ORMDL表达遗传学的基本问题， 来自哮喘的全基因组关联研究。单核苷酸多态性（SNP）邻近的 ORMDL同种型之一ORMDL3的基因与以下风险强烈且一致相关： 儿童哮喘和ORMDL3表达升高。尽管基于人口的研究 有趣和暗示，对这种现象的详细研究由于无法 直接测试这些SNP对ORMDL3表达的影响。无法直接测量 这些SNPs影响ORMDL3表达的程度，也不是在什么细胞类型和条件下，这种升高 表达发生。在这里，我们建议使用新兴的Crispr/Cas9基因组编辑技术来产生 配对细胞系，在哮喘相关细胞类型中，最多含有风险或非风险基因型 显著的风险相关SNP。这些将被用来检查的程度，细胞类型，和刺激特异性， 由风险等位基因诱导的ORMDL3表达的升高以及这如何影响鞘脂代谢。

Sphingolipids are a diverse but metabolically related group of lipids with unique functional and physical properties that are critical for cell signaling and membrane architecture. Misregulation of particular sphingolipids has been implicated in such diverse diseases as cancer, inflammatory disease, and cardiovascular disease. In particular, as outlined below, genome-wide association studies have strongly implicated the subject of this proposal, the sphingolipid metabolic regulator ORMDL, in the risk for childhood asthma. The overall level of cellular sphingolipid is determined by the initiating, and rate limiting enzyme in the biosynthetic pathway, serine palmitoyltransferase (SPT). Not surprisingly, SPT activity is homeostatically controlled-i.e. SPT activity is strongly inhibited by elevated levels of cellular sphingolipid. We have demonstrated that this homeostatic control is mediated by a set of small membrane proteins found in the endoplasmic reticulum, the ORMDLs. The goal of the studies proposed here is to establish the mechanism by which the ORMDLs mediate SPT inhibition in response to elevated sphingolipid levels and to begin to uncover how those mechanisms impact on risk for asthma. These studies will answer basic questions concerning how ORMDLs sense cellular sphingolipid levels and how changes in sphingolipid levels are transmitted through ORMDL to affect SPT activity. We will test the hypothesis that ceramide directly binds ORMDL to trigger SPT regulation. We will determine the structural elements in ORMDL that are essential for mediating sphingolipid regulation of SPT, will use a recently constructed cell-free system to quantitate the levels of sphingolipid sensed by the ORMDL system, and will test the hypothesis that elevation of sphingolipid induces changes in ORMDL topology. Finally we will construct a reconstituted system with purified ORMDL and SPT to test the hypothesis that the ORMDLs and SPT are the only required components of this regulatory system. In addition we propose to address an essential question concerning the genetics of ORMDL expression that has emerged from genome-wide association studies in asthma. Single nucleotide polymorphisms (SNPs) adjacent to the gene of one of the ORMDL isoforms, ORMDL3, are strongly and consistently associated with the risk for childhood asthma and with elevated expression of ORMDL3. Although the population-based studies are intriguing and suggestive, a detailed examination of this phenomenon has been hamstrung by the inability to directly test the effect of these SNPs on ORMDL3 expression. It has not been possible to directly measure the degree to which these SNPs affect ORMDL3 expression nor in what cell types and conditions this elevated expression occurs. Here we propose to use emerging Crispr/Cas9 genome editing technology to produce paired cell lines, in asthma-relevant cell types, containing either a risk or non-risk genotype at the most prominent risk-associated SNP. These will be used to examine the extent, cell type, and stimuli specificity of elevation of ORMDL3 expression induced by the risk allele and how this affects sphingolipid metabolism.